Recent studies employing a modified Western blot protocol have demonstrated that the 14-3-3 protein is the most useful biochemical marker in human cerebrospinal fluid (CSF) for Creutzfeldt-Jakob disease (CJD). A quantitative three-layer enzyme-linked immunosorbent assay (ELISA) has been developed that reliably detects 14-3-3 in CSF within the range of 2.1 to 135 ng/ml. The intra-assay and inter-assay coefficients of variation for the ELISA are 0.03 and 0.10, respectively. In a blinded case control study of 200 patient samples, the mean 14-3-3 concentration in definite CJD patints (n=39) was significantly higher (student's t-test, p<0.0001) than in those afflicted with other neurological disorders (n=109). Selecting an optimal cutoff value of 8.34 ng/ml, the ELISA demonstrated a sensitivity of 95% and a specificity of 97%. False positive results were observed in three patients afflicted with vasculitis, glioma, and senile-dementia of unknown etiology, respectively. These results are consistent with those studies which utilized a 14-3-3 Western blot technique. We conclude that the 14-3-3 ELISA provides an important test for the premortem diagnosis of CJD.